The mitochondrion of P. falciparum is stripped of many of the typical functions of mitochondria, yet retains pathways essential to the parasite survival. The remaining mitochondrial functions are proving to be an exceptionally productive set of targets for antimalarial drug discovery. One approach to overcoming drug resistance is to design new drugs such that the fitness cost of resistance restricts the ability of mutant parasites to survive combination therapy or to persist in the absence of selection. Through whole organism high-throughput screening we have discovered many new chemotypes that appear to act on mitochondrial targets. We propose to take a chemical biology approach to the mitochondrion and develop these into a suite of reagents useful in characterizing mitochondrial function by pinpointing mechanisms of action and resistance for a range of chemotypes that act against the mitochondrion. We will study the effects of the resistance mutations on the structure and function of the targets and the impact of those mutations and consequent biochemical changes on parasite growth and fitness in vitro. We will select resistance to a range of chemical inhibitors that appear to target the mitochondrial ETC, focusing on DHODH. Using an approach that has proven highly productive, we will characterize the resistant mutants to identify the target of the chemical inhibitor. We propose to conduct selections in sufficient depth and using a sufficiently diverse range of chemistry to sample the range of possible targets and mechanisms affecting the mitochondrial ETC, and to explore the range of resistance mutations to DHODH inhibitors intensively, focusing on the compound advancing to clinical testing. We will screen previously identified DHODH inhibitor screening hits for those active against the resistant parasites. We will map the resistance mutations of mutants newly isolated and assess the biological consequences by measuring the effects of the mutations on the enzyme, on mitochondrial function and on the growth of the organism.